1. Field of the Invention
Paratuberculosis poses a significant economic and health problem worldwide, especially in the cattle industry..sup.1,2 Mycobacterium avium subspecies paratuberculosis.sup.3 (M. paratuberculosis ) is the etiologic agent of paratuberculosis (Johne's disease), a chronic granulomatous enteritis of both domestic and wild ruminants. This organism is an intracellular pathogen that replicates within macrophage of both the gastrointestinal tract and associated lymphatic tissues..sup.4 The disease can be transmitted in utero, to nursing calves, or via infected fecal contamination of food. Diagnosis of subclinical paratuberculosis is problematic because infection progresses slowly and infected animals often do not show signs of the disease for years. Once the disease is established in a herd, there is no cure. Annual economic losses to the dairy industry are in the billions of dollars worldwide, primarily as a result of reduced milk production, decreased reproductive efficiency, and death.
In humans, M. paratuberculosis has been isolated from patients with Crohn's disease, a chronic enteritis with clinical symptoms similar to animals with paratuberculosis..sup.5,6 M. paratuberculosis has been implicated as a possible cause of Crohn's disease, however, the etiology of this disease remains unknown.
This invention relates to a species-specific genetic target element useful for identifying M. paratuberculosis and for distinguishing this organism from related bacteria by various diagnostic techniques. Probes and primer sets are disclosed for detecting target sequence in laboratory and clinical samples containing M. paratuberculosis.
2. Description of the Prior Art
Cattle shed M. paratuberculosis in their feces during the subclinical and clinical stages of infection. Currently, the most sensitive test available for subclinical paratuberculosis requires a prolonged 8-12 week fecal culturing of the organism. Existing immunological diagnostic tests are rapid but have disadvantages resulting from poor specificity of the antigens used in the assays.
Nucleic acid diagnostic methodology is used as a rapid and sensitive way to identify specific species of mycobacteria..sup.7,8,9 Some mycobacterial species are genetically very closely related to M. paratuberculosis according to DNA-DNA hybridization analysis..sup.10 Genome homology ranging from 50% to nearly 100% has been reported between the ATCC 19698 reference strain of M. paratuberculosis and species of the Mycobacterium avium complex (MAC) which includes the incompletely separated Mycobacterium avium (subspecies avium [M. avium] and subspecies silvaticum [M. silvaticum] and Mycobacterium intracellulare as well as other strains not assigned to either species..sup.11,12,13 M. paratuberculosis DNA is also related to DNA of other mycobacteria, such as Mycobacterium bovis, Mycobacterium leprae, and M. tuberculosis..sup.14,15 The high percentage of genetic relatedness of M. paratuberculosis with other mycobacterial species requires the cloning, sequencing, and characterization of unique genetic markers (genetic elements or genes) to differentiate these closely related species. Species-specific genetic markers are useful tools for the development of new molecular diagnostic tests.
Only two species-specific genetic elements have been identified in the M. paratuberculosis genome..sup.16,17 DNA probes derived from these genetic elements have been used to detect M. paratuberculosis infection. One genetic element, IS900, is a 1.45 kbp insertion element found at approximately 20 copies per chromosome..sup.16 Similar insertion elements, which have sequences related to IS900, have been identified in closely related mycobacteria, such as M. avium (IS901).sup.18,19 and M. silvaticum (IS902)..sup.20 The now commercially available IS900 DNA diagnostic kit (IDEXX Corp.), which was used in studies conducted in a M. paratuberculosis control program, yields an 89% specificity and a 13% sensitivity..sup.21
The other M. paratuberculosis species-specific genetic element that has been identified, F57, is a 620 bp DNA fragment not related to any known sequence, including the IS900 insertion element..sup.17 Southern hybridization analysis using the F57 fragment suggests that this genetic element is single-copy in the M. paratuberculosis genome. The F57 genetic element is currently being used as a diagnostic tool to identify M. paratuberculosis infection in both cattle and humans (patients with Crohn's disease)..sup.10,17 However, the cloning and sequencing of additional M. paratuberculosis-specific genetic elements or genes is needed to improve or develop new rapid and sensitive nucleic acid diagnostic tests for the differentiation of paratuberculosis infection.
Currently, there is a need for an accurate, rapid and reliable detection of M. paratuberculosis infection.